Switzer WM, Heneine W; National Conference on Human Retroviruses and Related Infections.
Program Abstr Second Natl Conf Hum Retrovir Relat Infect Natl Conf Hum Retrovir Relat Infect 2nd 1995 Wash DC. 1995 Jan 29-Feb 2; 62.
Centers for Disease Control Prevention, Atlanta, GA.
Intact nef genes in SIV mac have been shown to be important for maintaining high viral loads and for the development of simian AIDS. Nucleotide substitutions that introduce premature stop codons, or deletions in the SIV Nef reading frame lead to low viral replication and absence of disease in SIV-infected macaques. Currently, full-length or truncated Nef proteins are predicted from the nucleotide sequences of the SIV nef genes. However, analysis of the Nef open reading frames (ORF) by sequencing becomes labor intensive when a large number of animals are studied and when multiple quasispecies from each animal are examined. We evaluated the use of an in vitro transcription and translation assay (TT) to rapidly identify full-length or truncated SIV Nef proteins synthesized from PCR amplified nef genes. A total of 48 nef genes cloned from 14 SIV smm- infected monkeys were screened for open reading frames using the TT assay and the results were compared with DNA sequencing. Of these 48 genes, 20 had an intact Nef ORF and 28 had premature stop codons. All 20 nef genes with intact ORFs produced full-length proteins, while truncated proteins of different sizes were synthesized from all 28 nef genes with premature stop codons. By being rapid and reliable this technique should facilitate the examination of Nef ORFs in studies with large sample sizes.
Publication Types:
Keywords:
- Animals
- Base Sequence
- Biological Assay
- Gene Products, nef
- Genes, nef
- In Vitro
- NEF protein, SIV
- Open Reading Frames
- Protein Biosynthesis
- Simian Acquired Immunodeficiency Syndrome
- Simian immunodeficiency virus
- Transcription, Genetic
- Viral Load
- Viral Regulatory and Accessory Proteins
- analysis
- diagnosis
- genetics
Other ID:
UI: 102213017
From Meeting Abstracts